The present invention relates to a group of novel benzisoxazole derivatives, to a method for the preparation of these compounds, and to pharmaceutical compositions containing one or more of these compounds as an active component.
It has surprisingly been found that the compounds and salts thereof of the formula (I) 
wherein
(R1), represents 0, 1 or 2 substituents, which can be the same or different, from the group C1-3-alkyl or alkoxy, halogen, trifluoromethyl, nitro, amino, mono- or dialkyl (C1-2)-amino, sulfonyl-(C1-3)alkyl or -alkoxy, sulfonyl trifluoromethyl, sulfonyl amino, and sulfonyl mono- or dialkyl (C1-2)-amino,
X is O, S, NH or NCH3,
Y represents CH2 or (CH2)2 
(R2)m represents 0, 1, or 2 substituents, which can be the same or different, from the group methyl and ethyl, or (R2)m is a methylene bridge or ethylene bridge,
A is a group xe2x80x94CH2xe2x80x94(CRH)pxe2x80x94 wherein R is hydrogen or methyl and p is 0 or 1, and
B represents 2- or 3-indolyl or 2-benzimidazolyl, which groups may be substituted at carbon with 1 or 2 substituents from the group C-1-3-alkyl or alkoxy, halogen, trifluoromethyl, nitro, amino, mono- or dialkyl (C1-2)amino, sulfonyl-(C1-3)alkyl or -alkoxy, sulfonyl trifluoromethyl, sulfonyl amino, and sulfonyl mono- or dialkyl (C1-2)amino,
are potent and selective antagonists of the dopamine D4-receptor.
Compounds having formula (I) wherein A is the group CH2, Y is CH2, X is O, NH or NCH3 and m and n are 0, and B has the above meaning, and salts thereof are preferred.
Due to the potent and selective D4 antagonistic activity the compounds according to the invention are suitable for use in the treatment of psychiatric disorders such as psychosis, anxiety, depression, attention deficits and memory disorders, neurological disorders such as Parkinson""s disease and ischaemia and other CNS-diseases involving dopaminergic neurotransmission.
The affinity of the compounds of the invention for dopamine D4 receptors was determined using CHO-K1 cells which are stably transfected to express the human recombinant dopamine receptor, D4.2 subtype (Van Tol et al, Nature 350, 610, 1991) and using [3H]-Spiperone as the ligand. After incubation of a freshly prepared cellmembrane preparation with the [3H]-ligand, with or without addition of compounds of the invention, separation of bound and free ligand was performed by filtration over glassfiberfilters (Research Biochemicals International protocol, Catalog No. D-177). Radioactivity on the filter was measured by liquid scintillation counting. Results are expressed as IC50 values and transformed into inhibitory constants (Ki).
The dopamine D4 antagonistic activity of compounds of the invention was determined by functional studies using CHO-K1 cells stably expressing the human dopamine D4.4 receptor (Van Tol et al, Nature 358, 149, 1992).These cells were fitted with a construct encoding a truncated form of alkaline phosphatase, causing it to get secreted by the cells. Expression of this secretable alkaline phosphatase (SeAP) is under direct control of cellular cyclic AMP (Berger et al, Gene, 66, 1, 1988). SeAP measurements were done with p-nitrophenylphosphate (pNPP) as the substrate using colorimetric readout at 450 nm. Dopamine D4 antagonist activity was determined by co-incubation of cells with prostaglandin PGE1 (1 xcexcM) and quinpirole (1 xcexcM), with or without addition of compounds of the invention, for receptor-mediated stimulation of adenylate cyclase and for maximal dopamine D4 receptor-mediated suppression, respectively. The antagonistic effect of compounds of the invention against agonist dependant attenuation of dopamine D4 receptor mediated SeAP formation was quantified, yielding estimates of intrinsic activity and potency (pA2 values). Clozapine and spiperone were used as reference dopamine antagonists.
Absence of dopamine D4 agonistic activity was confirmed using the same assay, but leaving out the standard dopamine D4 agonist quinpirole, by determination of the concentration-dependant attenuation of the dopamine D4 receptor mediated SeAP formation by compounds of the invention.
Dopamine D4 antagonist properties and absence of dopamine D4 agonist properties of selected compounds of the invention were further confirmed using radioactive measurements of cAMP formation according to Salomon et al. (Anal Biochem, 58, 541, 1974) as modified by Weiss et al. (J Neurochem 45, 869, 1985).
The selectivity of the compounds of the invention with regard to the dopamine D2 receptor, was determined by measuring the affinity for dopamine D2 receptors using rat brain homogenates and [3H]-Spiperone as the ligand (Leysen et al, Biochem Pharmacol 27, 307, 1978). After incubation of a freshly prepared cellmembrane preparation with the [3H]-ligand, with or without addition of compounds of the invention, separation of bound and free ligand was performed by filtration over glassfiberfilters. Radioactivity on the filter was measured by liquid scintillation counting. Results are expressed as IC50 values and transformed into inhibitory constants (Ki).
The dopamine D2 (ant)agonistic activity of compounds of the invention was determined by functional studies based on radioactive measurements of cAMP formation according to Salomon et al. (Anal Biochem, 58, 541, 1974), as modified by Weiss et al. (J Neurochem, 45, 869, 1985), using CHO cells, stably expressing human dopamine D2L receptors (Grandy et al, Proc Natl Acad Sci USA, 86, 9762, 1989). Suitable acids with which the compounds can form pharmaceutically acceptable acid addition salts are for example hydrochloric acid, sulphuric acid, phosphoric acid, nitric acid, and organic acids such as citric acid, fumaric acid, maleic acid, tartaric acid, acetic acid, benzoic acid, p-toluene sulphonic acid, methane sulphonic acid and naphthalene sulphonic acid.
The compounds of the invention can be brought into forms suitable for administration by means of usual processes using auxiliary substances and/or liquid or solid carrier materials.
The compounds of the invention having formula (I) can be obtained according to methods known for the synthesis of structurally related compounds.
A suitable synthesis for the compounds according to the present invention is the following:
Step 1
Reaction of a compound having formula (II) 
with a compound of the formula (Ill) 
This reaction is carried out in a polar aprotic solvent such as dimethylformamide in the presence of an equivalent amount of a base such as sodiumhydride at 20-120xc2x0 C. The protecting benzyl group is then removed from the obtained product.
Step 2
When B is the group 2- or 3-indolyl, the thus obtained deprotected compound having formula (IV) 
is reacted with an optionally substituted 2- or 3-indolyl carboxylic acid derivative of the formula B-Axe2x80x2xe2x80x94COOH, wherein Axe2x80x2 is the group -(CRH)pxe2x80x94, wherein R is hydrogen or methyl and p has the value 0 or 1. This reaction is carried out in the presence of an equivalent amount of 1,1xe2x80x2-carbonyldiimidazole in an aprotic solvent such as tetrahydrofuran.
Step 3
The keto group in the obtained compound of the formula (V) 
is reduced to CH2 in a manner known per se, e.g. by means of an excess of sodium borohydride in the presence of acetic acid in a solvent such as dimethoxyethane under an atmosphere of nitrogen to give the desired compound having formula (I).
To prepare a compound having formula (I) wherein B is the group 2-benzimidazolyl, the compound having formula (IV) is reacted with an optionally substituted 2-halomethyl benzimidazole derivative of the formula Bxe2x80x94Axe2x80x94Z, wherein A has the above meaning and Z is Cl or Br. This reaction is carried out in the presence of a base such as triethylamine in a polar aprotic solvent such as acetonitrile at 20-80xc2x0 C.